1. Field of the Invention
The present invention is directed to purified EG III cellulase enzyme isolated from Trichoderma longibrachiatum and its amino acid sequence in both secreted and non-secreted forms. The present invention is further directed to a DNA sequence that encodes the EG III cellulase enzyme. The present invention further relates to methods of isolating purified and highly enriched EG III cellulase obtained from Trichoderma spp. or genetically modified strains of Trichoderma spp.
2. State of the Art
Cellulases are known in the art as enzymes that hydrolyze cellulose (.beta.-1,4-glucan linkages) thereby resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. While cellulases are produced (expressed) in fungi, bacteria and the like, cellulase produced by certain fungi, and in particular by the fungal genus Trichoderma spp. (especially Trichoderma longibrachiatum), have been given the most attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures.
In regard to the above, Wood et al, "Methods in Enzymology", 160, 25, pages 234 et seq (1988), disclose that complete fungal cellulase systems comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (EC 3.2.1.91) ("CBH"), endoglucanases (EC 3.2.1.4) ("EG"), and .beta.-glucosidases (EC 3.2.1.21) ("BG"). The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. CBHs and EGs have been isolated from a variety of fungal sources.
The complete cellulase system comprising CBH, EG and BG components is required to efficiently convert crystalline cellulose to glucose. Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose. Moreover, a synergistic relationship is observed between the cellulase components, particularly if they are of different classification.
On the other hand, cellulases and components thereof, used either singularly or in combination, are also known in the art to be useful in detergent compositions, as a softening agent, and to improve the feel of cotton fabrics, and the like. However, there is a problem with using the EG I and EG II components derived from Trichoderma spp., and especially Trichoderma longibrachiatum, in detergent compositions. Specifically, such components have their maximal activity at acidic pHs whereas most laundry detergent compositions are formulated for use at neutral or alkaline (pH&gt;7 to about 10) conditions. While it is disclosed in U.S. Ser. No. 07/668,640 that the use of one or more acidic endoglucanase components of Trichoderma longibrachiatum in detergent compositions will provide improvements in softening, color retention/restoration and feel to cotton-containing fabrics even when treated under alkaline conditions, U.S. Ser. No. 07/707,647 is directed to the discovery that the EG III component of Trichoderma spp. provides for superior and unexpected advantages in detergent compositions as compared to the EG I and EG II components of Trichoderma longibrachiatum.
In addition to its use in laundry detergents, EG III cellulase can be used in a pre-washing step in the appropriate solution at an intermediate pH where sufficient activity exists to provide desired improvements in color retention/restoration, softening and feel as disclosed in U.S. Ser. No. 07/707,647 filed May 30, 1991 and incorporated herein by reference.
EG III cellulase has a further use in the stonewashing process of colored fabrics wherein redeposition of a colorant onto the fabric may be reduced by employing purified EG III. This process is disclosed in U.S. Ser. No. 07/954,113 filed Sep. 30, 1992 and incorporated herein by reference.
Additionally, it is further contemplated that the high activity under neutral to alkaline conditions of EG III cellulase would be beneficial in textile processes for treating cotton-containing fabrics (see U.S. Ser. Nos. 7/677,385 and 07/678,865 which are incorporated herein by reference in their entirety) as well as in silage and/or composting processes.
Thus, it has become of increasing interest to isolate EG III in purified form or to create a Trichoderma strain which secretes a cellulase product enriched for EG III for commercial use. Others in the field have described the purification of low molecular weight endoglucanases from Trichoderma (Shoemaker et al (1981) Trends in the Biology of Fermentations for Fuels and Chemicals (Hollaender, Rabson, Rogers, Pietro, Valentine and Wolfe, Eds.), Plenum Publishing Corp., New York; Hakansson et al, (1978) Biochim. Biophys. Acta 524:385-392; Beldman et al (1985) Eur. J Biochem. 146:301-308; Ulker and Sprey (1990) FE MS Microbiol. Lett. 69:215-220 and Sprey and Ulker (1992) FEMS Microbiol. Lett. 92:253-258). However, it is not possible to determine which, if any, represent the same protein as the EG III described herein. For example, the protein isolated by Ulker and Sprey (1990) was determined to have an arginine at its amino terminus. However, the DNA sequence of the EG III encoding gene reported herein would predict that EG III has a glutamine residue at its amino terminus.
In light of the various applications of EG III, the present invention is directed to the complete characterization of EG III, i.e, the amino acid sequence and DNA sequence encoding EG III, purified from a fungal cellulase composition. The full characterization of EG III cellulase described herein will provide a cost-effective commercially available EG III cellulase product through genetic engineering and/or large scale protein purification procedures.